This invention relates to calcitonin receptor binding reagents which are capable of complexing with metal ions, including radioactive metal ions, and to labeled embodiments of such reagents for use in imaging sites in a mammalian body or for use in therapy, particularly for use in cancer therapy.
Great strides have been made over the past 60 years in reducing long-term mortality trends for some types of cancer, such as stomach cancer. However, during the same period, mortality trends for other cancers have remained stable or increased. For example, lung cancer is the most frequent cancer worldwide, representing the leading cause of cancer mortality among men and women. Breast cancer is the commonest cancer among women and the second leading cause of cancer mortality in women, and ovarian cancer mortality rates are increasing in some countries. childhood and adult lymphatic cancers, such as leukemias and non-Hodgkin""s lymphomas, also continue to represent significant causes of cancer mortality. Early diagnosis and effective treatment remains a goal for all of these cancers.
Several years ago, site directed diagnosis and therapy were proposed, to allow in vivo targeting of particular sites of disease within an animal""s body. In general, site-directed diagnosis or therapy employs a targeting moiety, such as an antibody specific for the disease site or for the organism which caused the disease, coupled to a label in the case of a diagnostic agent or to a cytotoxic agent in the case of a therapeutic agent. A very large body of literature exists relating to radiolabeling antibodies or antibody fragments for diagnostic imaging purposes. Similarly, a number of site directed therapeutic agents employing monoclonal antibodies and a variety of radioisotopes have been proposed over the years, e.g., as set forth in U.S. Pat. Nos. 4,454,106; 4,472,509; 4,828,991; 5,246,691; 5,355,394; and 5,641,471; in EP 429624; EP 585986; WO 90/15625, and the like. Such antibody-based agents produce side effects related to the immune responses of the treated animal to the antibody, even if antibody fragments or humanized antibodies are employed as the targeting moiety.
The disadvantages of antibody-based site-directed diagnostic and therapeutic agents can be avoided when targeting moieties having lower molecular weights, such as receptor-specific peptides or small molecules are employed. However, coupling of a peptide or small molecule to a label or cytotoxic agent, while retaining the compound""s receptor specificity, can be technically difficult. Methods for radiolabeling peptides and other small molecules which preserve the ability of the compound to bind specifically to a receptor are disclosed in commonly owned U.S. Pat. Nos. 5,225,180; 5,405,597; 5,443,815; 5,508,020; 5,552,525; 5,561,220; 5,620,675; 5,645,815; 5,654,272; 5,711,931; 5,716,596; 5,720,934; and 5,736,122; in abandoned U.S. patent application Ser. No. 07/955,466; and in WO92/13572, WO93/10747, WO93/17719, WO93/21962, WO93/23085, WO93/25244, WO94/00489, WO94/07918, and WO94/28942. The methods disclosed in these patents and publications are particularly suitable for manufacture of site-directed diagnostic imaging agents. Commonly assigned U.S. Pat. Nos. 5,620,675; 5,716,596; WO 94/00489; WO 95/03330; WO 95/00553; WO 95/31221; and WO 96/04308 disclose somatostatin peptide analogs which may be used for site-directed radiotherapy. Commonly assigned WO 95/33497 discloses somatostatin analogs, gpIIb/IIIa receptor-binding peptides, and leukocyte-binding peptides which may be used for site-directed radiodiagnosis or radiotherapy. Commonly assigned WO 96/30055 discloses vasoactive intestinal peptide (VIP) receptor-binding peptides which may be used for site-directed radiodiagnosis or radiotherapy.
Tumor cells often occasionally express or overexpress a particular receptor or receptor subtype, as indicated by receptor binding studies. In some types of cancer, tumor cell markers can change as the disease progresses, possibly reflecting the stage of the disease and thus the patient""s prognosis. The kind of receptor that a tumor cell expresses can be characteristic of the tumor""s etiology and can thus provide a relatively specific marker for the tumor. For example, radiolabeled somatostatin analogs have been shown to bind specifically to neuroendocrine tumors, melanomas, lung cancer, and certain breast cancers. One such analog, 111In-OCTREOSCAN, has received marketing approval for use in imaging neuroendocrine tumors. A second radiolabeled somatostatin analog, 99mTc-Depreotide, has Completed Phase III clinical trials for use in imaging lung cancers. 123I-vasoactive intestinal peptide has been shown to target adenocarcinomas of the colon and stomach.
Calcitonin (CT) is a 32 amino acid peptide secreted from the thyroid in response to elevated serum calcium levels. Calcitonin has a number of biological effects, which are mediated by calcitonin receptors present on the surfaces of cells in the target organ. High affinity receptors for CT have been identified in bone, kidney, lung, and central nervous system. In bone, CT inhibits bone resorption by osteoclasts; in kidney, CT increases excretion of calcium ions; and in the central nervous system, the peptide induces analgesia, gastric acid secretion, and appetite inhibition. Small amounts of CT have been administered to animals and humans without toxic effects, and salmon CT is used clinically to treat such bone disorders as Paget""s disease, hypercalcemia of malignancy, and osteoporosis. Intravenously-administered calcitonin clears the blood rapidly and is excreted primarily in urine. The major sites of localization for administered CT are kidney, liver and the epiphyses of the long bones.
Circulating CT levels are considered to be a marker for some types or stages of cancer, for example, medullary thyroid carcinoma, small-cell lung cancer, carcinoids, breast cancer, and gastrointestinal cancer. High affinity CT receptors have been identified in lymphoid cells, human lung cancer cell lines, human breast cancer cell lines, and in primary breast cancer tissue. Findlay et al. (1981) Biochem. J. 196: 513-520 reports that CT receptors are overexpressed in certain breast, lung, ovarian and lymphoma cancer cell lines.
The amino acid sequences of CT from several species (human, salmon and eel) are set forth below:
hCT CGNLSTCMLGTYTQDFNKFHTFPQTAIGVG.AP.amide {SEQ ID NO.:1)
sCT CSNLSTCVLGKLSQELHKLQTYPRTNTGSG.TP.amide (SEQ ID NO.:2)
eCT CSNLSTCVLGKLSQELHKLQTYPRTDVGAGTP.amide (SEQ ID NO.:3)
(where single-letter abbreviations for amino acids can be found in Zubay, Biochemistry 2d ed., 1988, MacMillan Publishing: New York, p. 33, and where the underlined amino acids between the two cysteine residues in the amino terminal portion of the peptide represent a disulfide bond). Among species, nine residues, including the carboxyl-terminal proline amide and the disulfide-bonded cysteine residues at positions 1 and 7 are conserved. The salmon and eel CTs are more potent than human CT both in vitro and in vivo.
CT peptide analogs have been developed in which the chemically-labile disulfide is replaced with stable carbon-carbon linkages formed between 2-aminosuberic acid, as described in U.S. Pat. No. 4,086,221. CT analogs in which the amino terminal, midregion, or carboxyl terminal portion of the molecule are deleted demonstrate only weak binding to CT receptors. Many amino acid substitutions may be made between residues 8 and 22 of the CT molecule to generate biologically active CT analogs. Some CT analogs with only minimal sequence homology to any natural form of CT have biological activity similar to that of salmon CT. Truncated CT peptide derivatives (such as Cbz-LHKLQY-OMe, SEQ ID NO.: 12) also retain substantial receptor binding activity.
Since tumors may express or overexpress different receptors, a variety of radiodiagnostic and radiotherapeutic agents are needed to afford optimal diagnostic and therapeutic modalities against cancer.
CT receptors on cell surfaces of lung and ovarian adenocarcinoma, breast cancers and lymphomas can be exploited as markers to locate, identify, and treat such tumors in vivo. The present inventors have for the first time developed small, synthetic compounds, including CT-derived peptides, possessing the capacity for high-affinity binding to CT receptors and favorable pharmacokinetics, thereby permitting efficient in vivo localization of diagnostic and therapeutic agents at tumor sites. The reagents of the invention provide the basis of rapid, cost-effective, non-invasive diagnostic imaging procedures useful for tumor detection, disease staging and evaluation of metastatic spread of tumors characterized by expression or overexpression of CT receptors. The reagents of the invention also provide the basis for assessment of therapeutic effectiveness of other treatment modalities, for example, by localization of CT receptor-expressing tumor cells following surgery, radiation therapy or chemotherapy. The reagents of the invention may also be used as targeting moieties for site-directed radiotherapy.
The invention provides CT receptor binding reagents comprising CT receptor binding compounds, preferably CT peptides, CT derivatives, or CT analogues, which are covalently linked to a radiometal chelator. The CT receptor binding compounds employed in the reagents of the invention have a molecular weight of less than about 10,000 daltons. In some embodiments, the reagents of the invention are characterized as peptides, by virtue of the presence of a peptide linkage either in the CT receptor binding portion of the reagent or by virtue of the presence of a peptide linkage in the radiometal chelator. The reagents of the invention have a CT receptor binding affinity that is not less than about one-tenth the affinity of radioiodinated native CT for said receptor, when compared in a standardized assay such as the assays described in Example 4 below. In preferred embodiments, the reagents of the invention have a CT receptor binding affinity equal to or greater than native CT or radioiodinated species of native CT for said receptor, when compared in said standardized assay.
The radiopharmaceuticals of the invention may be employed as site specific diagnostic or therapeutic agents. When labeled with technetium-99m, iodine-123, and iodine-13 1, the reagents of the invention may be employed as scintigraphic imaging agents. When labeled with a magnetic, paramagnetic, supermagnetic, or superparamagnetic metal, the reagents of the invention may be employed as magnetic resonance contrast agents. When labeled with a cytotoxic radionuclides, the reagents of the invention may be used for site-directed radiotherapy. The invention also provides pharmaceutical compositions comprising the radiolabeled CT receptor-binding compounds of the invention and a pharmaceutically acceptable carrier. Methods for making the CT receptor binding reagents of the invention and radiolabeled embodiments thereof are also provided.
The invention also provides kits for preparing radiolabeled CT receptor binding compounds from the reagents of the invention. The kits of the invention comprise a sealed vial containing a predetermined quantity of a reagent of the invention and optionally a sufficient amount of a reducing agent to radiolabel the reagent.
This invention provides methods for using the radiolabeled CT receptor-binding reagents of the invention diagnostically and therapeutically. In one embodiment, methods are provided for using reagents of the invention, in labeled form, for imaging sites within a mammalian body by obtaining in vivo images. These methods comprise the steps of administering an effective diagnostic amount of labeled reagents of the invention and detecting the label localized at the site within the mammalian body.
The invention also provides methods for alleviating diseases characterized by expression or overexpression of CT receptors, comprising the step of administering a therapeutically effective amount of a radiolabeled CT receptor-binding reagents of the invention to the animal.
Other aspects and advantages of the present invention are apparent in the following more detailed description of preferred embodiments set forth below.
The patent and scientific literature referenced herein establish the knowledge available to those with skill in the art. The issued U.S. patents and allowed applications are hereby incorporated by reference.
The present invention provides CT receptor binding reagents useful in the preparation of CT receptor binding pharmaceutical agents for diagnosis and therapy. For the purposes of this invention, the term xe2x80x9cCT receptor binding compoundxe2x80x9d is intended to encompass naturally-occurring CT, fragments of CT, analogues of CT, and derivatives of CT that specifically bind to the CT receptor expressed in a variety of cell types recognized by those with skill in the art. Compounds designed to mimic the receptor-binding properties of CT are also included in this definition and encompassed by the invention.
For the purposes of this invention, the term xe2x80x9cCT receptor binding affinityxe2x80x9d is intended to mean binding affinity as measured by any methods known to those of skill in the art, including, inter alia, those methods which measure binding affinity by a dissociation constant, an inhibition constant or an IC50 value.
The term xe2x80x9chaving a CT receptor binding affinity of at least one-tenth the affinity of radioiodinated CT for said receptorxe2x80x9d is intended to mean that the dissociation constant (Kd) of the reagent is not more than ten times the Kd of radioiodinated CT as measured in a CT receptor direct binding or competitive inhibition assay, or that the or inhibition constant (Ki) or IC50 of the reagent is not more than 10 times that of radioiodinated CT, as measured in a CT receptor competitive inhibition assay.
The term xe2x80x9chaving a CT receptor binding affinity equal to or greater than native CT or radioiodinated species of CT for said CT receptorxe2x80x9d is intended to encompass reagents having a dissociation constant (Kd) equal to or less than that of unlabeled CT or radioiodinated CT, as measured in a CT receptor direct binding or competitive inhibition assay. Alternatively, this term may be interpreted as encompassing reagents having an inhibition constant (Ki) or IC50 equal to or less than that of native CT or radioiodinated CT as measured in a CT receptor competitive inhibition assay.
In accordance with the invention, the reagents of the invention have a CT receptor binding affinity of at least ten times greater than the affinity of radioiodinated native CT for said receptor, when compared in a standardized assay as described below. In preferred embodiments, the reagents of the invention have a CT receptor binding affinity equal to or greater than native CT or radioiodinated species of native CT for said receptor, when compared in said standardized assay.
Dissociation constants or binding inhibition constants of the radiopharmaceuticals of the invention in relation to the CT receptor, as well as comparison of the affinity or avidity of such binding with binding of 125I-labeled CT itself, may be determined using known methods such as those set forth in Receptors, A Quantitative Approach, A. Levitzki, The Benjamin/Cummings Publishing Company (California, 1984). Preferably a standardized CT receptor binding assay, such as the assays set forth in Example 4 below, is employed to measure CT receptor binding affinity of the reagents of the invention.
In addition to the CT receptor binding compound, the reagents provided by this invention comprise a radiometal chelator which is covalently linked to the compound in such a way that the binding specificity of the compound for the CT receptor is not substantially altered. Any radiometal chelator may be covalently linked to a CT receptor binding compound to provide a reagent of the invention. For example, a CT receptor-binding compound, preferably a peptide, may be covalently linked to a chelator having the formula:
C(pgp)s-(aa)xe2x80x94C(pgp)s
where (pgp)s is hydrogen or a thiol protecting group and (aa) is any xcex1- or xcex2-amino acid not comprising a thiol group. In a preferred embodiment, the amino acid is glycine.
As another example, a radiometal chelator useful in the reagents of the invention comprises a single thiol-containing group of formula:
Axe2x80x94CZ(B)xe2x80x94{C(RaRb)}nxe2x80x94X
wherein A is H, HOOC, H2NOC, (peptide)-NHOC, (peptide)-OOC, Re2NCo, or Rd; B is H, SH or xe2x80x94NHRc, xe2x80x94N(Rc)-(peptide) or Rd; Z is H or Rd; X is SH or xe2x80x94NHRc, xe2x80x94N(Rc)-(peptide) or Rd; Ra, Rb, Rc and Rd are independently H or straight or branched chain or cyclic lower alkyl; n is 0, 1 or 2; Rc is C1-C4 alkyl, an amino acid or a peptide comprising 2 to about 10 amino acids; and: (1) where B is xe2x80x94NHRc or xe2x80x94N(Rc)-(peptide), X is SH and n is 1 or 2; (2) where X is xe2x80x94NHRc or xe2x80x94N(Rc)-(peptide), B is SH and n is 1 or 2; (3) where B is H or Rd, A is HOOC, H2NOC, (peptide)-NHOC, or (peptide)-OOC, X is SH and n is 0 or 1; (4) where A is H or Rd, then where B is SH, X is xe2x80x94NHRc or xe2x80x94N(Rc)-(peptide) and where X is SH, B is xe2x80x94NHRc or xe2x80x94N(Rc)-(peptide) and n is 1 or 2; (5) where X is H or Rd, A is HOOC, H2NOC, (peptide)-NHOC, or (peptide)-OOC and B is SH; (6) where Z is methyl, X is methyl, A is HOOC, H2NOC, (peptide)-NHOC, or (peptide)-OOC and B is SH and n is 0; and (7) where B is SH, X is not SH and where X is SH, B is not SH.
Preferred embodiments of this radiometal chelator have the chemical formula:
R1xe2x80x94Co-(amino acid)1-(amino acid)2xe2x80x94Z
wherein (amino acid)1 and (amino acid)2 are each independently any primary xcex1- or xcex2-amino acid that does not comprise a thiol group, Z is a thiol-containing moiety that is cysteine, homocysteine, isocysteine, penicillamine, 2-mercaptoethylamine or 3-mercaptopropylamine, and R1 is lower (C1-C4) alkyl, an amino acid or a peptide comprising 2 to 10 amino acids. When Z is cysteine, homocysteine, isocysteine or penicillamine, the carbonyl group of said moiety is covalently linked to a hydroxyl group, a NR3R4 group, wherein each of R3 and R4 are independently H or lower (C1-C4) alkyl, an amino acid or a peptide comprising 2 to 10 amino acids; or
Y-(amino acid)2-(amino acid)1-NHR2
wherein Y is a thiol-containing moiety that is cysteine, homocysteine, isocysteine, penicillamine, 2-mercaptoacetate or 3-mercaptopropionate, (amino acid)1 and (amino acid)2 are each independently any primary xcex1- or xcex2-amino acid that does not comprise a thiol group, and R2 is H or lower (C1-C4) alkyl, an amino acid or a peptide comprising 2 to 10 amino acids. When Y is cysteine, homocysteine, isocysteine or penicillamine, the amino group of said moiety is covalently linked to xe2x80x94H, an amino acid or a peptide comprising 2 to 10 amino acids.
In particular embodiments of this aspect of the invention, the formula of this radiometal chelator is selected from the group consisting of:
-(amino acid)1-(amino acid)2xe2x80x94Axe2x80x94CZ(B)xe2x80x94{C(R1R2)}nxe2x80x94X },
xe2x80x94Axe2x80x94CZ(B)xe2x80x94{C(R1R2)}nxe2x80x94X}-(amino acid)1-(amino acid)2,
-(a primary xcex1,xcfx89- or xcex2,xcfx89-diamino acid)-(amino acid)1xe2x80x94Axe2x80x94CZ(B)xe2x80x94{C(R1R2)}nxe2x80x94X}, and
xe2x80x94Axe2x80x94CZ(B)xe2x80x94{C(R1R2)})nxe2x80x94X}-(amino acid)1-(a primary xcex1,xcex2- or xcex1,xcex3-diamino acid) wherein (amino acid)1 and (amino acid)2 are each independently any naturally-occurring, modified, substituted or altered xcex1- or xcex2-amino acid not containing a thiol group; A is H, HOOC, H2NOC, (amino acid or peptide)-NHOC, (amino acid or peptide)-OOC or R4; B is H, SH or xe2x80x94NHR3, xe2x80x94N(R3)-(amino acid or peptide) or R4; Z is H or R4; X is SH or xe2x80x94NHR3, xe2x80x94N(R3)-(amino acid or peptide) or R4; R1, R2, R3 and R4 are independently H or straight or branched chain or cyclic lower alkyl; n is an integer that is either 0, 1 or 2; (peptide) is a peptide of 2 to about 10 amino acids; and: (1) where B is xe2x80x94NHR3 or xe2x80x94N(R3)-(amino acid or peptide), X is SH and n is 1 or 2; (2) where X is xe2x80x94NHR3 or xe2x80x94N(R3)-(amino acid or peptide), B is SH and n is 1 or 2; (3) where B is H or R4, A is HOOC, H2NOC, (amino acid or peptide)-NHOC, (amino acid or peptide)-OOC, X is SH and n is 0 or 1; (4) where A is H or R4, then where B is SH, X is xe2x80x94NHR3 or xe2x80x94N(R3)-(amino acid or peptide) and where X is SH, B is xe2x80x94NHR3 or xe2x80x94N(R3)-(amino acid or peptide) and n is 1 or 2; (5) where X is H or R4, A is HOOC, H2NOC, (amino acid or peptide)-NHOC, (amino acid or peptide)-OOC and B is SH; (6) where Z is methyl, X is methyl, A is HOOC, H2NOC, (amino acid or peptide)-NHOC, (amino acid or peptide)-OOC and B is SH and n is 0; and (7) where B is SH, X is not SH and where X is SH, B is not SH.
Specific preferred embodiments of this aspect of the invention include radiometal chelators having a formula selected from the group consisting of: -Gly-Gly-Cys-, Cys-Gly-Gly-, Gly-Gly-Cys-, -(xcex5-Lys)-Gly-Cys-, (xcex4-Orn)-Gly-Cys-, -(xcex3-Dab)-Gly-Cys-, -(xcex2-Dap)-Lys-Cys-, and -(xcex2-Dap)-Gly-Cys-. (In these formulae, it will be understood that xcex5-Lys represents a lysine residue in which the xcex5-amino group, rather than the typical xcex1-amino group, is covalently linked to the carboxyl group of the adjacent amino acid to form a peptide bond; xcex4-Orn represents an omithine residue in which the xcex4-amino group, rather than the typical xcex1-amino group, is covalently linked to the carboxyl group of the adjacent amino acid to form a peptide bond; xcex3-Dab represents a 2,4-diaminobutyric acid residue in which the y-amino group is covalently linked to the carboxyl group of the adjacent amino acid to form a peptide bond; and xcex2-Dap represents a 1,3-diaminopropionic acid residue in which the xcex2-amino group is covalently linked to the carboxyl group of the adjacent amino acid to form a peptide bond.)
In another embodiment, the radiometal chelator of the reagent of the invention is a bisamino-bisthiol chelator having the formula: 
wherein each R can be independently H, CH3 or C2H5; each (pgp)S can be independently a thiol protecting group or H; m, n and p are independently 2 or 3; A is linear or cyclic lower alkyl, aryl, heterocyclyl, combinations or substituted derivatives thereof; and X is peptide. Alternatively, the bisamino bisthiol chelator in this embodiment of the invention has the formula: 
wherein each R is independently H, CH3 or C2H5; m, n and p are independently 2 or 3; A is linear or cyclic lower alkyl, aryl, heterocyclyl, combinations or substituted derivatives thereof; V is H or CO-peptide; Rxe2x80x2 is H or peptide; provided that when V is H, Rxe2x80x2 is peptide and when Rxe2x80x2 is H, V is CO-peptide. For purposes of this invention, chelating moieties having these structures will be referred to as xe2x80x9cBATxe2x80x9d moieties.
Alternatively, the radiometal chelator used in the reagent of the invention may have a formula selected from the group consisting of:
diethylenetriaminepentaacetic acid (DTPA)
(HOOCCH2)2N(CR2)(CR2)N(CH2CoOH)(CR2)(CR2)N(CH2CoOH);
where each R is independently H, C1 to C4 alkyl, or aryl and one R is covalently linked to a bivalent linker;
ethylenediaminetetraacetic acid (EDTA)
(HOOCCH2)2N(CR2)(CR2)N(CH2CoOH);
where each R is independently H, C1 to C4 alkyl, or aryl and one R is covalently linked to a bivalent linker;
1,4,7,10-tetraazadodecanetetraacetic acid; 
where n is an integer that is 2 or 3 and where each R is independently H, C1 to C4 alkyl, or aryl and one R is covalently linked to the CT receptor binding compound, and desferrioxamine.
Most radiometals may be chelated to reagents of the invention comprising the above-mentioned radiometal chelators.
The reagents of the invention may also comprise a radiometal chelator selected from the group consisting of:
(i) a group having the formula: 
(ii) a group having the formula: 
wherein n, m and p are each integers that are independently 0 or 1; each Rxe2x80x2 is independently H, lower alkyl, C2-C4 hydroxyalkyl, or C2-C4 alkoxyalkyl, and each R is independently H or Rxe2x80x3, where Rxe2x80x3 is substituted or unsubstituted lower alkyl or phenyl not comprising a thiol group, and one R or Rxe2x80x2 is L, where L is a bivalent linker moiety linking the metal chelator to the targeting moiety and wherein when one Rxe2x80x2 is L, NRxe2x80x22 is an amine.
In preferred embodiments, L is a C1-C6 linear, branched chain or cyclic alkyl group, a carboxylic ester, a carboxamide, a sulfonamide, an ether, a thioether, an amine, an alkene, an alkyne, a 1,2-, 1,3- or 1,4-linked, optionally substituted, benzene ring, or an amino acid or peptide of 2 to about 10 amino acids, or combinations thereof.
In preferred embodiments, Rxe2x80x3 is a C1-C6 linear, branched or cyclic alkyl group; a xe2x80x94CqOCrxe2x80x94, xe2x80x94CqNHCrxe2x80x94 or xe2x80x94CqSCrxe2x80x94 group, where q and r are integers each independently 1 to 5 wherein the sum of q+r is not greater than 6; (C1-C6) alkyl-X, where X is a hydroxyl group, a substituted amine, a guanidine, an amidine, a substituted thiol group, or a carboxylic acid, ester, phosphate, or sulfate group; a phenyl group or a phenyl group substituted with a halogen, hydroxyl, substituted amine, guanidine, amidine, substituted thiol, ether, phosphate, or sulfate group; an indole group; a C1-C6 heterocyclic group containing 1 to 3 nitrogen, oxygen or sulfur atoms or combinations thereof.
In accordance with the invention, the radiometal chelator of the CT receptor-binding reagent may have the formula: 
wherein R1 and R2 are each independently H, lower alkyl, C2-C4 hydroxyalkyl, or C2-C4 alkoxyalkyl; R3, R4, R5 and R6 are independently H, substituted or unsubstituted lower alkyl or phenyl not comprising a thiol group; R7 and R8 are each independently H, lower alkyl, lower hydroxyalkyl or lower alkoxyalkyl; L is a bivalent linker group and Z is a CT peptide.
Additional preferred metal chelators of the invention include chelators of formula: 
wherein R1 and R2 are each independently H, lower alkyl, C2-C4 hydroxyalkyl, or C2-C4 alkoxyalkyl; R3, R4, R5 and R6 are independently H, substituted or unsubstituted lower alkyl or phenyl not comprising a thiol group, and one of R3, R4, R5 or R6 is Zxe2x80x94Lxe2x80x94HN(CH2)nxe2x80x94, where L is a bivalent linker group, Z is a targeting moiety, and n is an integer from 1 to 6; R7 and R8 are each independently H, lower alkyl, lower hydroxyalkyl or lower alkoxyalkyl; and X is an amino group, a substituted amino group or xe2x80x94NR1-Y, where Y is an amino acid, an amino acid amide, or a peptide comprising from 2 to 10 amino acids.
More preferred metal chelators of the invention include chelators having the formula: 
wherein R1 and R2 are each independently H, lower alkyl, lower hydroxyalkyl, or lower alkenylalkyl; R3 and R4 are independently H, substituted or unsubstituted lower alkyl or phenyl not comprising a thiol group; n is an integer from 1 to 6; L is a bivalent linker group; and Z is a CT peptide moiety.
Additional more preferred chelating moieties include chelators of formula: 
wherein L is a bivalent linker group and Z is a CT peptide moiety.
Most preferred chelating moieties of the invention include chelators having the following formulae:
(amino acid)1-(amino acid)2-cysteine-,
(amino acid)1-(amino acid)2-isocysteine-,
(amino acid)1-(amino acid)2-homocysteine-,
(amino acid)1-(amino acid)2-penicillamine-,
(amino acid)1-(amino acid)2-2-mercaptoethylamine-,
(amino acid)1-(amino acid)2-2-mercaptopropylamine-,
(amino acid)1-(amino acid)2-2-mercapto-2-methylpropylamine-,
(amino acid)1-(amino acid)2-3-mercaptopropylamine-,
wherein (amino acid) in a primary xcex1- or xcex2-amino acid not comprising a thiol group and wherein the chelator is attached to either a targeting moiety or a linker group via a covalent bond with the carboxyl terminus of the chelator or a side chain on one of the amino acid groups.
Most preferred chelators also include chelators of the above formula wherein (amino acid)1 is either an xcex1,xcfx89- or xcex2,xcfx89-amino acid wherein the xcex1- or xcex2-amino group is a free amine and the xcex1,xcfx89- or xcex2,xcfx89-amino acid is covalently linked via the xcfx89 amino group.
Other most preferred chelators include those selected from the group consisting of:
-cysteine-(amino acid)-(xcex1,xcex2- or xcex2,xcex3-diamino acid);
-isocysteine-(amino acid)-(xcex1,xcex2- or xcex2,xcex3-diamino acid);
-homocysteine-(amino acid)-(xcex1,xcex2- or xcex2,xcex3-diamino acid);
-penicillamine-(amino acid)-(xcex1,xcex2- or xcex2,xcex3-diamino acid);
2- mercaptoacetic acid-(amino acid)-(xcex1,xcex2- or xcex2,xcex3-diamino acid);
2- or 3-mercaptopropionic acid-(amino acid)-(xcex1,xcex2- or xcex2,xcex3-diamino acid);
2-mercapto-2-methylpropionic acid-(amino acid)-(xcex1,xcex2- or xcex2,xcex3-diamino acid);
wherein (amino acid) in a primary xcex1- or xcex2-amino acid not comprising a thiol group and wherein the chelator is attached to either a targeting moiety or a linker group via a covalent bond with the amino terminus of the chelator or a side chain on one of the amino acid groups.
Particularly preferred metal chelators are selected from the group consisting of: Gly-Gly-Cys-, Arg-Gly-Cys-, -(xcex5-Lys)-Gly-Cys-, -(xcex4-Orn)-Gly-Cys-, -(xcex3-Dab)-Gly-Cys-, -(xcex2-Dap)-Lys-Cys-. and -(xcex2-Dap)-Gly-Cys-. (In these formulae, the amino acid designations have the same meaning as is set forth above.)
An example of a radiometal chelator having structure III above is Gly-Gly-Cys-, wherein the chelating moiety has the structure: 
Chelating ligands having structure type VII form oxotechnetium complexes having the structure: 
An example of radiometal chelators having structure type V as shown above is Lys-(xcfx89-peptide)-Gly-Cys.amide which forms a chelator of structure: 
Chelating ligands having structure type IX form oxotechnetium complexes having the structure: 
An example of a reagent of the invention comprising a radiometal chelator having structure II as shown above is (targeting moiety)-Cys-Gly-xcex1,xcex2-diaminopropionamide which forms a chelator of structure: 
Radiodiagnostic agents having structure type XI form oxotechnetium complexes having the structure: 
In the radiometal chelators and CT receptor binding reagents provided by the invention that contain a thiol covalently linked to a thiol protecting group {(pgp)S}, the thiol-protecting groups may be the same or different and may be but are not limited to:
xe2x80x94CH2-aryl (aryl is phenyl or alkyl or alkyloxy substituted phenyl);
xe2x80x94CH-(aryl)2, (aryl is phenyl or alkyl or alkyloxy substituted phenyl);
xe2x80x94C-(aryl)3, (aryl is phenyl or alkyl or alkyloxy substituted phenyl);
xe2x80x94CH2-(4-methoxyphenyl);
xe2x80x94CH-(4-pyridyl)(phenyl)2;
xe2x80x94C(CH3)3 
-9-phenylfluorenyl;
xe2x80x94CH2NHCoR (R is unsubstituted or substituted alkyl or aryl);
xe2x80x94CH2xe2x80x94NHCoOR (R is unsubstituted or substituted alkyl or aryl);
xe2x80x94CoNHR (R is unsubstituted or substituted alkyl or aryl);
xe2x80x94CH2xe2x80x94Sxe2x80x94CH2-phenyl
Preferred protecting groups have the formula xe2x80x94CH2xe2x80x94NHCoR wherein R is a lower alkyl having between 1 and 8 carbon atoms, phenyl or phenyl-substituted with lower alkyl, hydroxyl, lower alkoxy, carboxy, or lower alkoxycarbonyl. The most preferred protecting group is an acetamidomethyl group.
When the reagent of the of the invention comprises a CT receptor binding compound which is a peptide, the peptide preferably comprises the amino acid sequence:
CH2Co.SNLSTXxe2x80x94 (SEQ ID NO.:10)
wherein X is selected from the group consisting of a cysteine residue, a homocysteine residue, and a homohomocysteine residue. Alternatively, the peptide may comprise the amino acid sequence:
CH2Co.X1NLSTX2xe2x80x94 (SEQ ID NO.:11)
wherein X1 is selected from the group consisting of an alanine residue, a glycine residue, and a serine residue; and X2 is selected from the group consisting of a cysteine residue, a homocysteine residue, and a homohomocysteine residue. Such peptides include naturally-occurring human CT and CT peptide analogs such as those which are specifically embodied in the amino acid sequences set forth below: CH2CO.SNLST.Hhc.VLGKLSCELHKLQTYPRTNTGSGTP.amide; (SEQ ID NO.:5) CH2CO.SNLST.Hcy.VLGKLSCELHKLQTYPRTNTGSGTP.amide; (SEQ ID No.:6) CH2CO.SNLST.Cys.VLGKLSCELHKLQTYPRTNTGSGTP.amide; (SEQ ID NO.:7) and SNLST.Asu.VLGKLSCELHKLQTYPRTNTGSGTP.amide (SEQ ID NO.:8)
Particularly preferred embodiments of the reagents of the invention include: CH2CO.SNLST.Hhc.VLGKLSC(BAT)ELHKLQTYPRTNTGSGTP.amide (SEQ ID NO.:4) CH2CO.SNLST.Hhc.VLGKLSQELHKLQTYPRTNTGSGTP(xcex5-K)GC.amide, (SEQ ID NO.:13) CH2CO.SNLST.Hhc.VLGKLSC(CH2CO.GGCK.amide)ELHKLQTYPRTNTGSGTP.amide, (SEQ ID NO.:14) CH2CO.SNLST.Hhc.VLGKLSC(Ch2CO.(xcex2-Dap)KCK.amide)ELHKLQTYPRTNTGSGTP.amide, (SEQ ID NO.:15) CH2CO.SNLST.Hhc.VLGKLSC(CH2CO.(xcex5-K)GCE.amide)ELHKLQTYPRTNTGSGTP.amide, (SEQ ID NO.:16) CH2CO.SNLST.Hcy.VLGKLSC(CH2CO.GGCK.amide)ELHKLQTYPRTNTGSGTP.amide, (SEQ ID NO.:17) CH2CO.SNLST.Hcy.VLGKLSC(CH2CO.(xcex2-Dap)KCK.amide)ELHKLQTYPRTNTGSGTP.amide, (SEQ ID NO.:18) CH2CO.SNLST.Hcy.VLGKLSC(CH2CO.(xcex5-K)GCE.amide)ELHKLQTYPRTNTGSGTP.amide, (SEQ ID NO.:19) CH2CO.SNLST.Cys.VLGKLSC(CH2CO.GGCK.amide)ELHKLQTYPRTNTGSGTP.amide, (SEQ ID NO.:20) CH2CO.SNLST.Cys.VLGKLSC(CH2CO.(xcex2-Dap)KCK.amide)ELHKLQTYPRTNTGSGTP.amide, (SEQ ID NO.:21) CH2CO.SNLST.Cys.VLGKLSC(CH2CO.(xcex5-K)GCE.amide)ELHKLQTYPRTNTGSGTP.amide, (SEQ ID NO.:22) SNLST.Asu.VLGKLSC(CH2CO.(xcex2-Dap)KCK.amide)ELHKLQTYPRTNTGSGTP.amide, (SEQ ID NO.:23)and SNLST.Asu.VLGKLSC(CH2CO.(xcex2-Dap)KCK.amide)ELHKLQTYPRTDVGAGTP.amide, (SEQ ID NO.:24).
All naturally-occurring amino acids are abbreviated using standard abbreviations (which can be found in G. Zubay, Biochemistry (2d. ed.), 1988 (MacMillen Publishing: New York) p.33). For the purposes of this invention, the naturally-occurring amino acids are characterized as lipophilic (alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, proline, tryptophan and valine, as well as S-alkylated derivatives of cysteine), hydrophilic (asparagine, glutamine, threonine, serine), acidic (glutamic acid and aspartic acid), basic (arginine, histidine and lysine). xcex5-K, xcex4-Orn, xcex3-Dab and xcex2-Dap have the meanings set forth above. (BAT) represents N6,N9-bis(2-mercapto-2-methyl-propyl)-6,9-diazanonanoic acid; K.(BAT) and Lys.(BAT) represent the amino acid lysine, acylated at the xcex5-amino group on the amino acid sidechain to (BAT); C(BAT) and Cys(BAT) represent S-(N6,N9-bis(2-mercapto-2methylpropyl)-6,9--diazanonan-1-yl)cysteine; (BAM) is (N1,N4-bis(2-mercapto-2-methylpropyl)-1,4,10-triazadecane; (BAT-BM) is N-{2-(Nxe2x80x2,Nxe2x80x2-bis(2-maleimidoethyl)aminoethyl}-N9-(t-butoxycarbonyl)-N6,N9-bis(2-methyl-2-triphenylmethylthiopropyl)-6,9-diazanonanamide; (BAT-BS) is N-{2-(Nxe2x80x2,Nxe2x80x2-bis(2-succinimidoethyl)aminoethyl)-N6,N9-bis(2-mercapto-2-methylpropyl)-6,9-diazanonanamide; (BMH) is bis-maleimidohexane; (BSH) is bis-succinimidohexane; (BMME) is bis-maleimidomethylether; (BSEE) is bis-succinimidoethylether; (BMEE) is bis-maleimidoethylether; and (BSME) is bis-succinimidomethylether. As used herein, the following amino acids and amino acid analogues are intended to be represented by the following abbreviations: Acm is the sulfhydryl protecting group acetamidomethyl; Pen is penicillarnine; Aca is 6-aminocaproic acid; Hly is homolysine; Apc is L-{S-(3-aminopropyl)cysteine; FD is D-phenylalanine; WD is D-tryptophan; YD is D-tyrosine; Cpa is L-(4-chlorophenyl)alanine; Thp is 4-amino-tetrahydrothiopyran-4-carboxylic acid; D-Nal is D-2-naphthylalanine; Dpg is dipropylglycine; Nle is norleucine; Hcy is homocysteine; Hhc is homohomocysteine; Aib is aminoisobutyric acid; Nal is 2-naphthylalanine; Dxe2x80x94Nal is D-2-naphthylalanine; Ain is 2-aminoindan-2-carboxylic acid; Achxa is 4-amino-cyclohexylalanine; Amf is 4-aminomethyl-phenylalanine; Aec is S-(2-aminoethyl)cysteine; Apc is S-(3-aminopropyl)cysteine; Aes is O-(2-aminoethyl)serine; Aps is O-(3-aminopropyl)serine; Abu is 2-aminobutyric acid; Nva is norvaline; and Asu is 2-amino suberic acid, wherein the amino terminal amino acids of peptides containing an Asu residue are cyclized via an amide bond between the amino terminal amino group and the side chain carboxylic acid moiety of the Asu residue.
In accordance with the invention, CT receptor binding peptides may comprise one or more amino acid derivatives having a radiometal chelator linked to an amino acid sidechain. Preferably, the radiometal chelator is incorporated into the peptide at the carboxyl terminus of the CT receptor binding peptide. More preferably, the radiometal chelator is incorporated into the synthetic, CT receptor binding peptide at the sidechain sulfur atom of a cysteine corresponding to position 14 of the native peptide. Most preferably, the radiometal chelator is incorporated into a sidechain of an amino acid of the CT receptor binding peptide having the sequence
CH2CO.SNLST.Hhc.VLGKLSCELHKLQTYPRTNTGSGTP.amide. (SEQ ID NO.:9)
Most preferably, the radiometal chelator is incorporated into the CT receptor binding peptide at the sidechain sulfur atom of the cysteine at position 13 of the peptide depicted above (distinguished by the bold highlighting in said peptide).
Additional embodiments of the reagents of the invention comprise at least two synthetic CT receptor binding compounds, each compound being covalently linked to a radiometal chelator, and a polyvalent linker forming a covalent linkage selected from the group consisting of a linkage to each compound, a linkage to each chelator, and a linkage to one compound and to the chelator of the other compound. Additional permutations of this embodiment may also occur, in accordance with the invention. Polyvalent linkers suitable for use in this embodiment of the invention comprise at least two identical functional groups capable of covalently bonding to CT analogues, CT receptor binding compounds, CT peptides or radiometal chelators, or capable of binding both to a CT receptor binding compound and to a radiometal chelator. Preferred functional groups include, without limitation, primary amines, secondary amines, hydroxyl groups, carboxylic acid groups or thiol-reactive groups. In preferred embodiments, the polyvalent linkers comprise bis-succinimidylmethylether (BSME), bis-succinimidylethylether (BSEE), 4-(2,2-dimethylacetyl)benzoic acid (DMBA), N-{2-(Nxe2x80x2,Nxe2x80x2-bis(2-succinimido-ethyl)aminoethyl)}-N6,N9-bis(2-methyl-2-mercapto-propyl)-6,9-diazanonanamide (BAT-BS), tris(succinimidylethyl)amine (TSEA), bis-succinimidohexane (BSH), 4-(O-CH2Co-Gly-Gly-Cys.amide)-2-methylpropiophenone (ETAC), tris(acetamidoethyl)amine, bis-acetamidomethyl ether, bis-acetamidoethyl ether, xcex1,xcex5-bis-acetyllysine, lysine and 1,8-bis-acetamido-3,6-dioxa-octane, or derivatives thereof.
CT receptor binding compounds provided by the present invention can be chemically synthesized in vitro using any suitable synthetic method. Preferably, CT peptides may be synthesized in accordance with the invention using recombinant methods. More preferably, CT peptides, CT peptide derivatives, and CT peptide analogues can generally advantageously be prepared in accordance with the present invention using a peptide synthesizer. The CT peptides of this invention are preferably synthesized by covalently linking the radiometal chelator to the peptide during chemical synthesis in vitro, using techniques well known to those with skill in the art such as solid phase peptide synthesis. In this manner, radiometal chelators may be incorporated into the peptide in a site-selective fashion at virtually any position in the peptide thereby avoiding a decrease in the affinity and specificity of the peptide for the CT receptor.
In accordance with the invention, CT receptor binding peptides are prepared having a protected thiol-containing amino acid, typically a cysteine residue, incorporated into the peptide. Following cleavage of the peptide from the synthetic resin and cyclization of the amino terminal residues, the protected thiol group is deprotected and elaborated with a prosthetic group containing a radiometal chelator and a thiol-reactive group.
As set forth above, the CT receptor binding reagents of the invention are capable of being radiolabeled to provide radiodiagnostic or radiotherapeutic agents. An exemplary radiodiagnostic application using the radiolabeled reagents of the invention is scintigraphic imaging, wherein the location and extent of CT receptor-bearing tumors may be determined. The term xe2x80x9cscintigraphic imaging agentxe2x80x9d as used herein is meant to encompass a radiolabeled reagent capable of being detected with a radioactivity detecting means (including but not limited to a gamma-camera or a scintillation detector probe). In the radiotherapeutic embodiments of the invention, CT receptor binding reagents are labeled with a cytotoxic radionuclides and are useful in the treatment of diseases or other ailments in animals, preferably humans, such diseases or ailments being characterized by expression or overexpression of CT receptors. CT-related diseases or ailments include but are not limited to breast cancer, ovarian cancer, lung cancer, lymphoma and other diseases characterized by the growth of malignant or benign tumors capable of binding CT receptor binding compounds, CT or derivatives or analogues thereof via the expression of CT receptors on the cell surface of cells comprising such tumors.
Any radiometal may be complexed with the reagents of the invention to provide a radiodiagnostic or radiotherapeutic agent. For example, the reagents of the invention may be radiolabeled with technetium-99m, iodine-125, or iodine-123 to provide a scintigraphic imaging agent. This invention also provides CT receptor-binding reagents capable of forming a complex with a magnetic, paramagnetic, supermagnetic, or superparamagnetic metal atom, ion or particle. The CT receptor binding reagents of the invention can also advantageously be radiolabeled with a cytotoxic radioisotope selected from the group consisting of scandium-47, copper-67, gallium-72, yttrium-90, tin-117m, iodine-125, iodine-131, samarium-153, gadolinium-159, dysprosium-165, holmium-166, ytterbium-175, lutetium-177, rhenium-186, rhenium-188, astatine-211, bismuth-212, and bismuth-213 to provide a radiotherapeutic agent.
When the reagents of the invention are used to form a complex of radioactive technetium or rhenium, the technetium complex, preferably a salt of technetium-99m pertechnetate, or rhenium in the form of perrhenate, is reacted with the reagent in the presence of a reducing agent. Preferred reducing agents are dithionite, stannous and ferrous ions; the most preferred reducing agent is stannous chloride. Alternatively, the complex may be formed by reacting a reagent of this invention with a pre-formed labile complex of technetium or rhenium and another compound known as a transfer ligand. This process is known as ligand exchange and is well known to those skilled in the art. The labile complex may be formed using such transfer ligands as tartrate, citrate, gluconate or mannitol, for example. Among the technetium-99m pertechnetate and rhenium salts useful with the present invention are included the alkali metal salts such as the sodium salt, or ammonium salts or lower alkyl ammonium salts.
The invention is also embodied in a kit for preparing radiometal-labeled reagents for use as radiopharmaceuticals. The kit of the invention comprises a sealed vial containing a predetermined quantity of the CT receptor binding reagent, and optionally, when the radiometal is technetium-99m, rhenium-186, or rhenium-188, a reducing agent. For example, an appropriate quantity of a reagent of the invention is introduced into a vial containing a reducing agent, such as stannous chloride, in an amount sufficient to label the reagent with technetium-99m, rhenium-186 or rhenium-188. An appropriate amount of a transfer ligand as described (such as tartrate, citrate, gluconate, glucoheptanate or mannitol, for example) can also be included in the kit. The kit may also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, buffers, preservatives and the like. The components of the kit may be in liquid, frozen or dry form. In a preferred embodiment, kit components are provided in lyophilized form. Technetium-99m, rhenium-186 and rhenium-188 labeled radiopharmaceuticals according to the present invention may be prepared by the addition of an appropriate amount of technetium-99m, rhenium-186 or rhenium-188, or radionuclide complexes thereof, into the vials and reaction under conditions described in the Examples below.
The kit of the invention may be embodied in a form suitable for diagnostic imaging or as a therapeutic agent using a radioisotope of iodine, including iodine-123 and iodine-131, and preferably iodine-123. In this embodiment, the kit comprises a sealed vial containing a predetermined quantity of a CT receptor binding reagent capable of being radiolabeled with an iodine isotope. CT receptor binding reagents suitable for use in this embodiment include CT itself, a CT derivative, a CT analogue, CT mimetics and CT peptidomimetics that specifically bind to the CT receptor. When peptide and peptidomimetic CT receptor binding reagents are employed in this embodiment, a tyrosine residue in the reagent may be radioiodinated. Such a tyrosine residue may occur naturally in the peptide or peptidomimetic, or the tyrosine residue may be added at a position in the peptide or peptidomimetic that does not disrupt binding of the reagent to CT receptors. Dose, sites and routes of administration, formulations and administered specific radioactivity using the kit of this embodiment are as described herein for technetium and rhenium-labeled reagents for scintigraphic and therapeutic uses.
The imaging agents provided by the invention have utility for tumor imaging, particularly for imaging primary and metastatic neoplastic sites characterized by neoplastic cells that express or overexpress CT receptors, and in particular such primary and especially metastatic breast, lung and ovarian tumor-derived cells that have been clinically recalcitrant to detection using conventional methodologies. The imaging reagents provided by the present invention can also be used for visualizing organs such as the kidney or bone for diagnosing disorders in these organs.
For diagnostic purposes, an effective diagnostic amount of the diagnostic or radiodiagnostic agent of the invention is administered, preferably intravenously. In radiodiagnostic embodiments, localization of the radiolabel is detected using conventional methodologies such as gamma scintigraphy. In non-radioactive diagnostic embodiments, localization of sites of accumulation of the paramagnetic metal4abeled diagnostic agents of the invention is achieved using magnetic resonance imaging methodologies.
In accordance with this invention, for scintigraphic imaging the technetium-99m labeled reagents of the invention are administered in a single unit injectable dose. The technetium-99m labeled reagents provided by the invention may be administered intravenously in any conventional medium for intravenous injection such as an aqueous saline medium, or in blood plasma medium. Generally, the unit dose to be administered has a radioactivity of about 0.01 mCi to about 100 mCi, preferably 1 mCi to 20 mCi. The solution to be injected at unit dosage is from about 0.01 mL to about 10 mL. After intravenous administration, imaging in vivo can take place in a matter of a few minutes. However, imaging can take place, if desired, in hours or even longer, after the radiolabeled reagent is injected into a patient. In most instances, a sufficient amount of the administered dose will accumulate in the area to be imaged within about 0.1 of an hour to permit the taking of scintiphotos. Any conventional method of scintigraphic imaging for diagnostic purposes can be utilized in accordance with this invention.
For the purposes of this invention, radiotherapy encompasses any therapeutic effect ranging from pain palliation to tumor ablation or remission of symptoms associated with the particular cancer being treated. When the reagents of the invention are used for therapeutic purposes, they are radiolabeled with an effective amount of a cytotoxic radioisotope. For this purpose, an amount of cytotoxic radioisotope from about 10 mCi to about 200 mCi may be administered via any suitable clinical route, preferably by intravenous injection.
In accordance with this invention, effective radiodiagnostic and radiotherapeutic agents may be identified as follows. Reagents of the invention comprising CT receptor binding compounds, including CT fragments, CT peptide analogues and CT derivatives, are synthesized using the methods of the invention and a radiometal chelator is covalently linked to the compound. The reagents are then complexed with a radiometal or a non-radioactive isotope having chelation characteristics similar to the desired radiometal, and CT receptor binding is then evaluated in in vitro competition binding assays as described herein using radioiodinated CT. As an example of this methodology, ReO is employed to evaluate the suitability of CT receptor-binding peptides for use as 99m-technetium radiolabeled scintigraphic imaging agents, as disclosed in Example 4 below.